JP3515793B2 - Antiallergic agent - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antiallergic agent. At present, acidic antiallergic agents such as sodium cromoglycate and tranilast and basic antiallergic agents represented by ketotifen fumarate and oxatomide are used for the treatment of allergic diseases. These drugs can sometimes have gastrointestinal and central side effects. Also, it is difficult to use in combination with steroids used to alleviate seizure symptoms, and careful use is required. From the viewpoints of safety and usage, development of safer and more effective drugs is desired. [0003] An object of the present invention is to provide an antiallergic agent derived from a natural product. Means for Solving the Problems The present inventors have found that an extract of a natural product, amateur, has an excellent antiallergic effect. Further, the search for the active ingredient revealed that tumberginols, which are an isocoumarin derivative and a benzylidenephthalide derivative, were active, and the present invention was achieved. That is, the present invention provides an antiallergic agent comprising an extract of sweet tea. The present invention also provides a compound of the formula: An antiallergic agent comprising a coumarin or benzylidenephthalide derivative represented by the formula:
The sweet tea used in the present invention is Saxifragaceae (Sxifragaceae) amateur (Hydrangeae macrophylla Seringe ver.thunber).
gii MAKINO) in which the leaves are fermented and adjusted, and there are few examples showing what kind of medicinal effect it has, and no examples showing any antiallergic effect. This time, it was determined that the sweet tea extract had an excellent antiallergic effect by performing a PCA reaction (passive skin anaphylaxis reaction) for detecting anaphylaxis, which is a type of immediate allergy, as described later. In the present invention, the above-mentioned sweet tea extract itself may be used as a pharmaceutically active ingredient. However, the present inventors have conducted further experiments and found the active ingredient. About sweet tea extract, soft extract, extract such as fluid extract and tincture,
It is prepared by an ordinary method according to the 12th revised Japanese Pharmacopoeia “General rules for preparations”. For example, the following is illustrated as a method for preparing a soft extract. Sweet tea is impregnated in the extraction solvent and filtered to obtain a filtrate. For the residue, the above-mentioned cold immersion and filtration are repeated two to three times. The obtained filtrates are combined, the extraction solvent is distilled off and concentrated to obtain an extract. It is preferable to stir occasionally during the cold immersion. [0008] Examples of the extraction solvent include water, alcohols such as methanol and ethanol, ethyl acetate, acetone, and mixtures thereof, and are preferably methanol or ethanol. The amount of the extraction solvent used is 2 to 10 parts by weight, preferably 4 to 5 parts by weight, based on 1 part by weight of the raw material. If the amount is less than 2 parts by weight, the extract cannot be sufficiently extracted, and if the amount is more than 10 parts by weight, the extraction effect is not improved, which is economically disadvantageous. The extraction temperature and time are 5 to 40 ° C., preferably 15 ° C. for cold immersion extraction.
At ２５25 ° C., for 1 to 5 days, preferably for 2 to 3 days. In the case of heat extraction, from 40 ° C. to near the boiling point of the extraction solvent, preferably at 70 to 80 ° C., for 30 minutes to 3 hours,
Preferably, it is 1-2 hours. In the above-mentioned extraction operation, the extraction may be carried out under normal pressure or reduced pressure, but it is preferable to carry out the concentration at a concentration of 40 ° C. or lower. The coumarin derivative may be used in the extraction mixture, but may be isolated if necessary. Isolation is performed by first extracting with an organic solvent such as a lower alkyl ketone, a lower alcohol, a lower fatty acid ester, or a lower fatty acid ether, and then subjecting the concentrate of this extract to column chromatography using silica gel, alumina or the like as an adsorbent. It can be done by doing. As lower alkyl ketones, for example, acetone and methyl ketone are used, as lower alcohols, for example, methanol and ethanol, and as lower fatty acid esters, for example, ethyl ether and isopropyl ether are used. Examples of extraction and separation from sweet tea are shown in Examples. Basically, the above-described extraction is appropriate, but other separation means may be used, or these separation means may be combined. As an example of the above isolated compound, Embedded image Embedded image The therapeutic agent of the present invention is used in the form of an oral solid preparation in combination with a commonly used pharmaceutical adjuvant. Such oral solid dosage forms include tablets,
Granules, capsules and the like can be mentioned. As an excipient,
Silicic anhydride, synthetic aluminum silicate, lactose, corn starch, crystalline cellulose and the like can be used, and carboxymethyl cellulose, pregelatinized starch and the like can be used as a binder. In addition, if necessary, a lubricant, a coating agent, a flavoring agent, a coloring amount, and the like can be contained. The pharmaceutical composition of the present invention is effective for treating and preventing allergies in humans. The dose of the pharmaceutical composition varies depending on factors such as symptoms and the constitution of the patient,
Generally, 0.5 extract powder per adult per day
It is suitable that the range of 0.1 to 3.0 g and the amount of the extract be 0.1 to 1.0 g. The amount of the active ingredient isocoumarin derivative and benzylidene phthalide derivative is 5 per adult per day.
A suitable amount is from 500 to 500 mg, preferably from 10 to 100 mg. According to the present invention, it is possible to provide a therapeutic agent which is extremely effective in preventing and treating allergic diseases as a new method for applying sweet tea. EXAMPLES Next, the antiallergic action of the present invention and preparation examples will be described. The present invention is not limited to these examples. Example 1 A sweet tea extract was prepared by fermenting the leaves of sweet tea.
Ethanol was added to 0 g, and the mixture was digested for 2 hours (water bath, 80 ° C.) and extracted. The same operation was repeated three times, the respective extracts were combined, and the solvent was distilled off under reduced pressure to obtain an ethanol extract. The yield was 5.8%. Example 2 PCA (Passivc Cu) involving immunoglobulin E (IgE)
Effect on Taneous Anaphylaxis Response Male Wistar rats (weight 140-160)
The back of g) was shaved, and a rat anti-DNP-roundworm protein (DNP-As) serum prepared separately, diluted with physiological saline, was sensitized by injecting 0.1 ml into the back skin. Forty-eight hours later, sweet tea extract (1000 mg / kg) was orally administered, and two hours later, DNP-As (0.75 mg protein) was dissolved in 0.5 ml of 1% Evan's blue-containing physiological saline and administered intravenously. After 30 minutes, the blood was exsanguinated and the dye leakage area was measured. Table 1 shows the results. [Table 1] The sweet tea extract-administered group significantly reduced the pigment leakage area compared to the control group. The inhibition rate of the PCA reaction was 31 to 54%. Example 3 Effect on PCA reaction involving immunoglobulin G (IgG) Male Hartley guinea pig (body weight 250-
280 g) of the back was shaved, and a rabbit anti-ovalbumin (EA) serum prepared separately, diluted with physiological saline, was sensitized by injecting 0.1 ml into the back skin. Two hours later, sweet tea extract (1000 mg / kg) was orally administered, and two hours later, ovalbumin (10 mg) was dissolved in 1 ml of 1% Evan's blue-containing physiological saline and administered intravenously. After 30 minutes, the blood was exsanguinated and the dye leakage area was measured. Table 2 shows the results. [Table 2] At the antiserum dilution factor of 5 × 10 ³ and 1 × 10 ⁴ , the group administered with the sweet tea extract was 20 to 30% higher than the control group.
2 × 10 ⁴ which suppressed dye leakage showed a significant suppression of about 60%. Formulation Example 1 Granules Granules having the following composition are prepared by a conventional method. Daily amount Sweet tea extract 0.2 g Corn starch 0.88 g Crystalline cellulose 0.8 g Sodium carboxymethyl cellulose 0.1 g Light anhydrous silicic acid 0.01 g Magnesium stearate 0.01 g Total 2.00 g Formulation example 2 Tablets Daily amount Sweet tea extract 0.50 g Corn starch 1.20 g Crystalline cellulose 0.50 g Lactose 1.48 g Light anhydrous silicic acid 0.80 g Magnesium stearate 0.02 g Total 4.50 g Formulation Example 3 Hard Capsule 1 Daily amount Sweet tea extract 0.10 g Corn starch 0.90 g Crystalline cellulose 0.50 g Hydroxypropyl cellulose 0.06 g Light anhydrous silicic acid 0.10 g Magnesium stearate 0.01 g Lactose 0.33 g Total 2.00 g Formulation example 4 Oral liquid preparation An oral liquid preparation having the following composition is prepared by a conventional method. Daily dose Sweet tea extract 100 mg Nikkor HCO 60 (solubiliser) 120 mg Pepper tincture (flavoring agent) 0.07 ml peppermint oil 0.002 ml sucrose (sweetener) 5.2 g ethanol (dissolution aid) 0.4 ml benzoic acid (Preservative) 24 mg Ethyl paraoxybenzoate 2 mg Purified water qs 60 ml Example 3 Extraction and separation from armature 5 kg of armature was heated and extracted with 5 times the amount of methanol, and a methanol extract (950 g) was obtained by a conventional method. Was. The methanol extract was dissolved in water and partitioned and extracted with ethyl acetate. Partition extraction was performed with ethyl acetate. The ethyl acetate transfer portion was subjected to silica gel chromatography "(chloroform / methanol) and (chloroform / methanol / water)", followed by reverse phase chromatography (60% methanol) and Sep.
By separating with hadex LH-20 (methanol),
Twelve compounds including six of the products of the present invention were obtained. Effect on Schults-Dale Reaction Passive sensitization was performed by intravenously administering 2 ml / kg of a separately prepared rabbit anti-ovalbumin serum to male Hartley guinea pigs weighing 250 to 350 g. Twenty-four hours later, the guinea pigs were sacrificed by exsanguination by carotid artery transection, and the trachea was immediately removed to remove attached fat and connective tissue. Next, the trachea was cut into a ring shape with a width of 2 mm, and the cartilage part on the opposite side of the muscle was cut open with scissors to connect three pieces to prepare a specimen. This was mounted on a Magnus apparatus filled with a nutrient solution (warmed at 37 ° C.) aerated with a 95% O ₂ -5% CO ₂ mixed gas with a load of 1 g. After stabilization, histamine hydrochloride was added to a final concentration of 10 ^-5 M. After washing with a nutrient solution and stabilizing, this operation was repeated once again, and the maximum contraction force obtained at this time was defined as H. Subsequently, after washing and stabilization, the test substance was added, and 10 minutes later, ovalbumin was added.
Challenge was performed by adding 1 μg / ml. The contraction force upon addition of the test substance was set to 0, and the maximum contraction force A generated by the challenge was determined. The test substance which was hardly soluble in water was dissolved in ethanol and added to the nutrient solution (final concentration of ethanol: 0.1%). The contractile force was normalized by setting the average value of A / H in the control group to 100 for each value, then the inhibition rate was calculated using the following formula, and the respective IC ₅₀ (50% Inhibitory Concentration) was determined. . The histamine contraction was performed by preparing a tracheal specimen of a non-sensitized guinea pig in the same manner as in the above-mentioned SD reaction experiment, and contracting it using 10 ⁻⁵ M histamine instead of ovalbumin. [Equation 1] [Table 3] As shown in Table 3, Tumberginol A, B,
C, D, E and F showed an inhibitory effect on SD response at low dose. The inhibitory effects of tubenbergols on histamine contraction were all weaker than the inhibitory effects on SD response. Effect on histamine release from mast cells Five Wistar male rats weighing 250-300 g were killed by exsanguination by carotid artery transection, and PBS (NaC
l: 154 mM KCl: 2.7 mM CaCl ₂ : 0.9 mM Glucose: 5.6 mM HEPES: 5 mM pH 7.4) 10
ml was injected and the abdomen was gently massaged for 2 minutes.
After laparotomy, ascites was collected and the peritoneal cavity was further washed with 10 ml of PBS. Next, after combining the obtained ascites fluid and the washing solution,
Centrifugation (120 g, 10 minutes, 4 ° C.) was performed to obtain a cell pellet. The cell pellet is washed with PBS and centrifuged (120 g
After repeating the operation twice for 10 minutes at 4 ° C), PBS 0.5m
l resuspended. The cell suspension was purified by density gradient centrifugation to obtain a mast cell fraction, and then PBS was added to adjust the concentration to 6 × 10 ⁻³ cells / ml, and the mast cell suspension (MC solution) was obtained. And After pre-incubating 1.8 ml of the MC solution at 37 ° C. for 15 minutes, 0.2 ml of a test substance solution (a solution dissolved in a small amount of dimethyl sulfoxide and dispersed in water) was added, and further incubated for 15 minutes. Continued. Next, as a histamine releasing substance, Compound (Compoun
d) Histamine was released by adding 0.22 ml of 48/80 (3 μg / ml) and incubating for 10 minutes. The released histamine concentration was measured using a fluorescence method. The histamine release inhibition rate of the test substance was calculated using the following formula, and the IC ₅₀ (50% Inhibitory Co
ncentration). [Mathematical formula-see original document] [Table 4] As shown in Table 4, Tumberginol A, B,
D, E and F suppressed histamine release from mast cells at low doses. Toxicity test 10 ddy male mice weighing 20 to 25 g were grouped into 10 mice,
Tumberginol A to F was orally administered at 3000 mg / kg, respectively, and observed for one week. No deaths were observed. Formulation Example 5 Granules A granule having the following composition is prepared by a conventional method. Daily dose Tumberginol A 0.05 g Corn starch 1.03 Crystalline cellulose 0.8 g Sodium carboxymethyl cellulose 0.1 Light anhydrous silicic acid 0.01 Magnesium stearate 0.01 Total 2.00 g Formulation example 6 Tablets A granule having the following composition is prepared by a conventional method. Daily dose Tumberginol B 0.05 g Corn starch 1.65 Microcrystalline cellulose 0.50 Lactose 1.48 Light anhydrous silicic acid 0.80 Magnesium stearate 0.02 Total 4.50 g Formulation example 7 Oral liquid formulation An oral liquid preparation having the following composition is produced by the method. Daily dose Tumberginol F 100 mg Nikkor HCO-60 (solubiliser) 120 mg Pepper tincture (flavoring agent) 0.07 ml peppermint oil 0.002 ml sucrose (sweetener) 5.2 g ethanol (dissolution aid) 0.4 ml benzoic Acid (preservative) 24mg Purified water qs 60ml

Continued on the front page (72) Inventor Hisashi Matsuda 1-1-30 Tamazuki, Chuo-ku, Osaka-shi, Osaka Inside Morishita Jintan Co., Ltd. Morishita Nittan Co., Ltd. (72) Inventor Hiroshi Shimoda 1-1-30 Tamazo, Chuo-ku, Osaka, Osaka Prefecture Morishita Nittan Co., Ltd. Within Morishita Jintan Co., Ltd. (56) References: Tomoko Tsuruga et al, Biological Active Constituents of Melaleuca leukad endron: Inhibitors of Insulated Histamine. Pharm m. Bull. 1991, Vol. 39, NO. 12, pp. 3276-3278 Hisako Kakegawa et al, Inhibitory Effects of Hydrangen ol Derivatives on the Activation of Hyaluronidase and Others, 1988. 5, pp. 385-389 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 A61K 31/365 BIOSIS (STN) CA (STN) JICST file (JOIS) MEDLINE (STN)

Claims (1)

(57) [Claims] [Claim 1] Formula: embedded image An antiallergic agent containing a benzylidenephthalide derivative represented by the formula: